This project is focused on the identification of physiologically critical functions of NF-kappaB proteins and their regulators. NF-kappaB is a family of related dimeric transcription factors that serve as primary intracellular mediators of signals that are generated during innate and adaptive immune responses. In addition, dysregulation of NF-kappaB contributes significantly to inflammatory and autoimmune diseases as well as a wide variety of tumors. To identify novel physiologic roles we make use of mouse models engineered to lack components of the NF-kappaB transcription factor family or their regulators. These mutant mice are challenged in vivo in various ways to reveal failed biologic responses. Once a defect is identified this provides the physiologic context in which to dissect out specific cellular and molecular contributions of NF-kappaB proteins or their regulators. NF-kappaB regulators of interest include the non-classical and non-inhibitory IkappaB protein Bcl-3, as well as components of certain signaling pathways that lead to activation of NF-kappaB. We analyze the mutant mice primarily for defects in immune system development, for impaired immune responses to pathogens as well as for failed responses to various stress- or disease-inducing agents. The ultimate goal is to identify critical physiologic processes that depend on NF-kappaB factors and their regulators in health and disease. We aim to identify the relevant cell types involved, the NF-kappaB-activating signaling pathways and the targets of NF-kappaB. We have shown previously that mice deficient in the activation of the alternative pathway for NF-kappaB in stromal cells have defects in secondary lymphoid organ architecture, in addition to defects in B cell development. The alternative pathway is activated by select members of the TNF receptor family, including the receptors for BAFF, RANK and lymphotoxin beta. Activation of the alternative pathway is mediated via processing of the NF-kappaB2 p100 precursor to p52, which leads to translocation of in particular RelB-containing NF-kappaB heterodimers into the nucleus. We have also previously demonstrated that mice deficient in the NF-kappaB regulatory protein Bcl-3 exhibit stromal cell-intrinsic defects in secondary lymphoid architecture as well, even though Bcl-3 does not have a role in the alternative pathway of NF-kappaB activation. We have since generated mice deficient in both NF-kappaB2 and Bcl-3. In FY 2008 we have now shown that these mutant mice develop a phenotype dramatically distinct from that seen in either single knockout. Unexpectedly, these compound mutant mice developed a fatal multi-organ inflammation shortly after birth. In FY 2008 we further determined that this severe autoimmune reaction was due to a failure to negatively select T cell in the thymus, which in turn was due to defects intrinsic to thymic stromal cells. Stromal medullary epithelial cells did not develop, which prevented the generation and display of autoantigens in the thymus, required for negative selection of autoreactive T cells. In another study we continue to investigate why the loss of NF-kappaB2 ameliorated various parameters of obesity-induced insulin resistance in these mutant mice, a condition which precedes diabetes type 2 in humans. A role for NF-kappaB2 was surprising in that only the classical pathway of NF-kappaB activation has been associated with insulin resistance; NF-kappaB2 is not part of this pathway.